Molecular interactions of medically important arthropod-borne viruses (arboviruses) with the mosquito, Aedes aegypti, are poorly described. Understanding these interactions is vital to developing novel arbovirus control strategies that reduce the mosquito's ability to transmit arboviruses in the field. Arboviruses such as flavi- and alphaviruses have been shown to act as targets for RNAi when replicating in the mosquito midgut. Arboviruses must adapt to the RNAi pathway to survive in the mosquito vector and the vector must adapt to arbovirus invasion. We hypothesize that the RNAi machinery in the mosquito midgut acts as a critical antiviral innate immune response that modulates the replication of arboviruses and therefore has an important role in vector competence. Previous experiments have shown that transient disruption of the RNAi pathway in Ae. aegypti can significantly alter arbovirus infection patterns. But we do not know how arboviruses overcome RNAi. We plan to manipulate the RNAi pathway in the midgut of transgenic mosquitoes to understand how RNAi modulates arbovirus infections in the mosquito. To generate transgenic mosquitoes efficiently we will use the phage phiC31 site-specific integrase system. Therefore, a recipient strain of Ae. aegypti, HWE, will be engineered that harbors the phiC31 attachment site (Specific Aim 1). Through site-specific transformation of the recipient strain we will generate transgenic Ae. aegypti lines that express (1) the FHVR1deltaB2-EGFP replicon as an indicator for RNAi suppression, (2) the FHV suppressor of RNAi, B2, or (3) three inverted repeat RNAs targeting components of the RNAi pathway such as dicer-2, R2D2, and argonaute-2 in the midgut (Specific Aim 2.). All transgenic mosquito lines will be infected with dengue, Sindbis, and Chickungunya viruses (Specific Aim 3). Indication of RNAi suppression in the midgut, virus replication efficiencies, virus tissue tropism, mosquito life span and fecundity will be evaluated. We expect to reveal which of the viruses actively suppresses RNAi in the mosquito midgut. We also expect to see changes in replication efficiency and tissue tropism of the viruses in RNAi-compromised mosquitoes and how the viruses affect the life cycle of Ae. aegypti when they are not controlled by the mosquito's RNAi response. The proposed research will help to reveal the impact of RNAi as an antiviral defense mechanism in the midgut of Ae. aegypti. [unreadable] [unreadable] [unreadable]